Visualization of introduced dna (void) in transit by in situ hybridization

ABSTRACT

The invention relates to a process for monitoring exogenous nucleic acid in transit, termed Visualization Of Introduced DNA (VOID). Once the nucleic acid has been introduced into a cell in a biological sample, the cells are fixed and permeabilized if necessary, then subjected to an in situ hybridization procedure in which the fixed cells are contacted with a probe which hybridizes to the exogenous nucleic acid. The exogenous nucleic acid, in transit, can thus be visualized. VOID may be used (a) to determine the efficiency of delivery of the nucleic acid into the nucleus; (b) to assess the risk associated with DNA delivery procedures where, by tracking the fate of the DNA, it can be determined whether too many of the exogenous DNA are delivered, which may lead to an undesired consequence; (c) to control the copy number of DNA delivery during the development of a transgenic product or a product containing an exogenous nucleic acid as the active ingredient; (d) to identify cells as having been transformed or transfected with an exogenous DNA, without the use of selection markers or reporters; (e) to identify molecular markers associated with transformation competency of a cell and identifying a cell that is competent to receive exogenous nucleic acid; (f) identifying, characterizing and producing cells competent to receive exogenous nucleic acid. VOID has been used successfully to identify the cellular protein VirD2-Interacting protein (VDI) as such a molecular marker. Thus a cell may be identified as being transformation-competent if and when it expresses VDI.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/368,524, filed Apr. 1, 2002, the content of which is hereinincorporated by reference.

FIELD OF INVENTION

The invention relates to a process for monitoring exogenous nucleic acidby in situ hybridization. The nucleic acid is monitored in transit onceit has been introduced into a cell.

BACKGROUND OF THE INVENTION

Recombinant DNA technology can be used for various applications in thebiomedical, agricultural, environmental, and industrial fields. Theseoften require gene or DNA delivery and transformation. DNA molecules canbe delivered into mammals as DNA vaccines. DNA molecules containinguseful genes can be applied as therapeutics or so-called gene therapy.Genetic modifications of animal, plant or microbial organisms based ontransgenic technology can lead to development of various high valueproducts.

To develop these gene-based or DNA-based products, it is crucial tomonitor the gene (DNA) delivery and transformation processes andefficiencies. Current techniques for this purpose are based on detectionof the expressed gene product of the DNA delivered. Therefore, marker orreporter genes are used for the detection of gene delivery andtransformation. To allow the marker or reporter gene to be expressed,appropriate promoters that can drive the expression of the deliveredgene have to be included. However, a promoter may not be functional incertain cell type(s), stage(s), and/or condition(s). The activity of apromoter in certain cell type(s), stage(s), and/or condition(s) may betoo weak for the detection. Alternatively, the activity may be so strongthat it can lead to the toxicity of a marker or reporter gene. Inaddition, enough time has to be given to the marker or reporter gene tobe expressed. This can considerably prolong the time needed to developDNA-based or gene-based products.

Inclusion of the marker or reporter gene(s) in a gene-based or DNA-basedproduct may also harm public acceptance of the product, because of thepotential effects of the marker or reporter gene(s) or its product(s) onthe environment and human health. Furthermore, detection of the markeror reporter protein cannot readily reveal the transgene copy numbers orlocations of the desired DNA, which are important for assessing theefficacy and safety of the material containing the desired DNA. This isbecause having too many copies of the transgene delivered into thetarget cells may lead to unwanted integration at vital sites of the hostchromosomes, which could be harmful to the host.

In situ hybridization is a technique which uses direct hybridization ofa DNA probe with DNA or RNA in biological structures, typicallypermeabilized cells, subcellular fractions, or fixed chromosomepreparations. The technique is often directed toward a target sequencein a double-stranded duplex nucleic acid, typically a DNA duplexassociated with a pathogen or with a selected sequence in viral or cellchromosomal DNA. A single-stranded labeled probe is annealed to thedenatured target duplex nucleic acid, and the structure is processed forvisualization of the annealed probe, thus allowing the location of theprobe within the target duplex nucleic acid to be determined.

In situ hybridization has been used to reveal morphological informationabout the localization of sequence-specific targets in fixed biologicalstructure. Specifically the method has been widely applied tochromosomal DNA, for mapping the location of specific gene sequences,and distances between known gene sequences, for studying chromosomaldistribution of satellite or repeated DNA, for examining nuclearorganization, for analyzing chromosomal aberrations, for localizing DNAdamage in single cells or tissue and for determining chromosome contentby flow cytometric analysis. Localization of integrated viral sequenceswithin host-cell chromosomes have been reported. The method has alsobeen used to study the position of chromosomes, by three-dimensionalreconstruction of sectioned nuclei, and by double in situ hybridizationwith mercurated and biotinylated probes, using digital image analysis tostudy interphase chromosome topography. In situ hybridization has alsobeen used to detect the presence of virus in host cells, as a diagnostictool.

Cheng L, Bucana C D, Wei Q. Fluorescence in situ hybridization methodfor measuring transfection efficiency. Biotechniques September 1996;21(3):486-91, states “We describe here the use of fluorescence in situhybridization (FISH) to measure the transfection efficiency of thetransient expression vector pCMVcat in lymphoblasts and fibroblasts.”Cheng et al. showed that, for transfection of pCMVcat by thediethylaminoethyl-dextran method, the transfection efficiency was about15 and 70 times greater in fibroblasts and lymphoblasts, respectively,when measured by FISH as compared to the efficiency measured bycotransfection with pCMV beta gal.

In Co D O, Borowski A H, Leung J D, van der Kaa J, Hengst S, PlatenburgG J, Pieper F R, Perez C F, Jirik F R, Drayer J I. Generation oftransgenic mice and germline transmission of a mammalian artificialchromosome introduced into embryos by pronuclear microinjection.Chromosome Res 2000;8(3):183-91, transgenic mice were generated bypronuclear microinjection of a murine satellite DNA-based artificialchromosome (SATAC). FISH analyses of metaphase chromosomes frommitogen-activated peripheral blood lymphocytes from both the founder andprogeny revealed that the SATAC was maintained as a discrete chromosomeand that it had not integrated into an endogenous chromosome.

Collas P, Alestrom P. Nuclear localization signals enhance germlinetransmission of a transgene in zebrafish. Transgenic Res Jul.1998;7(4):303-9 reported that cytoplasmic injection into zebrafish eggsof plasmid DNA complexed to nuclear localization signal (NLS) peptidesincreased nuclear uptake of transgene DNA early during embryodevelopment. It states that “Monitoring the dynamics of nuclear uptakeof DNA-NLS complexes by fluorescence in situ hybridization (FISH) ofinterphase nuclei indicates that NLS enhances both the proportion ofnuclei importing DNA during early embryo development, and the amount ofDNA imported by individual nuclei.”

SUMMARY OF THE INVENTION

The invention relates to the use of a modified in situ hybridizationprocedure to monitor the progress of introduced nucleic acid as it makesits way into the cell, through the cytoplasm and into the nucleus. Inthe past, in situ hybridization has been used to visualize introducedDNA at its endpoint; for example, DNA after it has integrated into achromosome, or episomal DNA once it has reached the point where it hasbecome replicated and/or has reached the nucleus and can be expressed(transcribed). The present invention relates to visualizing the nucleicacid in transit, prior to, or just as it reaches, its endpoint. Theprocess is useful for defining the optimum parameters for nucleic aciddelivery under a given set of conditions.

The process is termed Visualization Of Introduced DNA (VOID). Once thenucleic acid has been introduced into a cell in a biological sample, thecells are fixed and optionally permeabilized if necessary, thensubjected to an in situ hybridization procedure in which the fixed cellsare contacted with a probe which hybridizes to the exogenous nucleicacid. The exogenous nucleic acid, in transit, can thus be visualized.

Although the abbreviation “VOID” refers to DNA, the process is clearlyapplicable to any introduced nucleic acid including RNA.

In one embodiment, the nucleic acid is DNA. In another embodiment, thenucleic acid is DNA and is introduced into the cell by Agrobacterium.

In situ hybridization may be fluorescence in situ hybridization,radioactive in situ hybridization, or enzymatic in situ hybridization.

In one aspect, the process described above may be used to determine thenumber of exogenous nucleic acid in the cytoplasm or in the nucleus.

In another aspect, the process described above may be used to determinewhether the exogenous nucleic acid is in the cytoplasm or the nucleus.

In another aspect, the process described above may be used to determinethe length of time required for the exogenous nucleic acid to appear inthe cytoplasm.

In another aspect, the process described above may be used to determinehow long it takes for the exogenous nucleic acid to reach the nucleusfrom the cytoplasm.

In another aspect, the process described above may be used to determinethe efficiency of delivery of the nucleic acid into the nucleus. Thisprocess would further comprise the step of measuring the ratio of thenumber of the exogenous nucleic acid in the nucleus to the number of theexogenous nucleic acid in the cytoplasm.

In another aspect, the process described above may be used to assessrisk associated with introduction of the exogenous nucleic acid into thecell, including the risk associated with the use of a particular vehiclefor nucleic acid delivery such as a particular vector system. Thisprocess further comprises the step of determining the number ofexogenous nucleic acid in the cytoplasm and in the nucleus at differenttime intervals after the exogenous nucleic acid has been introduced. Theratio of exogenous nucleic acid in the nucleus to cytoplasm isdetermined at each interval. This allows VOID to predict, in accordancewith said ratio and number of exogenous nucleic acid introduced, therisk associated with introduction of the exogenous nucleic acid into thecell or with the use of a particular vehicle for nucleic acid delivery.

In another aspect, the process described above may be used to controlthe copy number of the exogenous nucleic acid introduced into the cell.

In another aspect, there is described a process for determining theproportion of cells competent to receive exogenous nucleic acid. Theprocess comprises: (a) introducing an exogenous nucleic acid to aportion of a population of cells; (b) monitoring the exogenous nucleicacid according to the process described above to determine the presenceof the exogenous nucleic acid in the cell; and (c) determining thenumber of cells in which the exogenous nucleic acid is present. Theproportion of cells which contain the exogenous nucleic acid as observedwith VOID reflects the proportion of cells competent to receive theexogenous nucleic acid.

In another aspect, VOID may be used to identify whether a cell containsan exogenous nucleic acid, without having to use a selection marker orreporter protein. The process comprises:(a) introducing the exogenousnucleic acid into the cell; and (b) monitoring the exogenous nucleicacid according to the process described above. Visualization of thenucleic acid in the cell indicates that the cell contains the exogenousnucleic acid.

In another aspect, VOID may be used to identify a molecular markerassociated with the competency of a cell to receive exogenous nucleicacid. The process comprises:(a) introducing an exogenous nucleic acid tothe cell; (b) monitoring the exogenous nucleic acid according to theprocess described above;(c) testing the fixed cells for binding of acellular antigen with an antibody. The antibody should be capable ofbinding to the antigen in the fixed and permeabilized cell; and (d)determining whether the antigen co-localizes with the exogenous nucleicacid in transit. Co-localization of the exogenous nucleic acid intransit with the antigen would indicate that the antigen is a molecularmarker associated with transformation competency.

In another aspect, VOID may be used to determine the optimum parametersfor obtaining a desired copy number of exogenous nucleic acid introducedinto the cell, the process comprising: (a) introducing an exogenousnucleic acid into a cell under a set of parameters; (b) monitoring theexogenous nucleic acid according to the process described above todetermine the number of exogenous nucleic acid in the cytoplasm or inthe nucleus at different time intervals after the nucleic acid has beenintroduced; and (c) determining the set of parameters under which theexogenous nucleic acid is delivered in the desired copy number into thecell. In one embodiment, at least one of the parameters is the length oftime in which the exogenous nucleic acid is in contact with the cell.

In another aspect, VOID may be used to identify a cell that is competentfor receiving exogenous nucleic acid, the process comprising monitoringthe exogenous nucleic acid according to the process described above forpresence of the exogenous nucleic acid in the cell.

In another aspect, VOID is used to identify a cell competent to receiveexogenous nucleic acid. The process comprises identifying expression ofa Sec3 protein in the cell. Sec3 expression would indicate that the cellis competent to receive exogenous nucleic acid. In one embodiment, thecell is a plant cell and the Sec3 protein is VirD2-Interacting protein(VDI).

In another aspect, there is described a process for producing cellscompetent to receive exogenous nucleic acid, the process comprising thestep of expressing Sec3 protein in the cell under control of aninducible promoter.

In another aspect, VOID is used to identify a cell competent to receiveexogenous nucleic acid. The process comprises the step of identifyingexpression of a component of Exocyst complex in the cell. Expression ofthe component indicates that the cell is competent to receive exogenousnucleic acid.

In another aspect there is described a kit for monitoring exogenousnucleic acid in transit, the nucleic acid having been introduced into acell. The kit comprises: (a) reagents for fixing the cells; (b) reagentsfor permeabilizing the fixed cells; (c) reagents for in situhybridization of a probe with the exogenous nucleic acid; and (d)instructions for using the reagents (a) to (c) to monitor the exogenousnucleic acid in transit.

In one embodiment, the processes described above are applied to plantcells. Such processes may further comprise the step of removing the cellwall.

DESCRIPTION OF THE DRAWINGS

Various embodiments of the invention are illustrated by way of thefigures described below. The figures are originally in color. Thevarious symbols are used to indicate the colored objects observed infigures which are not printed in color.

FIG. 1. Schematic presentation of relevant gene constructs in A.tumefaciens. The Ti plasmid contains the vir genes (virA-E and virG)that are required for the transfer of T-DNA harbored on pIG121-Hm (OhtaS. et al. 1990. Construction and expression in tobacco of aβ-Glucuronidase (GUS) reporter gene containing an intron within thecoding sequence. Plant Cell Physiol., 31(6), 805-813) and delineated bythe left (LB) and right border (RB). The T-DNA contains the GUS genedriven by the 35S promoter (P35s). The small arrows indicate the primersused to amplify PCR fragments, which were labeled as probes for VOID.Primers GUS-1 (5′-CGTCCTGTAGAAACC-3′; SEQ ID NO:2) and GUS-2(5′-ACGCACAGTTCATAG-3′; SEQ ID NO:3) were used to generate a 755-bpfragment, which can be used as the T-DNA probe to detect T-DNA insideplant cells. Primers BIN19-1 (5′-TTGCTCATGTTACCG-3′; SEQ ID NO:4) andBIN19-2 (5′-GCAGTTCCGCAAATA-3′; SEQ ID NO:5) were used to generate a757-bp fragment, which can be used as the vector backbone probe todetect the presence of the plasmid backbone. Primers Oligo-105(5′-GAAGAATTCGAACTTGACGCCGATACC-3′; SEQ ID NO:6) and Oligo-107(5′-AGGCTGCAGACATGCGTATTTTCG-3′; SEQ ID NO:7) were used to generate a677-bp fragment, which can be used as the aopB probe to detect thepresence of A. tumefaciens chromosomal DNA.

FIG. 2. The requirements for visualization of T-DNA inside plant cells.The BY2 cells were cocultivated with LBA4404(pIG121-Hm) (vir⁺) (panelsA, D and F), MX243(pIG121-Hm) (virB⁻) (panel B), WR1715(pIG121-Hm)(virD2⁻) (panel C), or MX358(pIG121-Hm) (virE2⁻) (panel E) for 1 day.They were then subjected to the VOID procedure using the T-DNA (panelsA, B, C and E), aopB (panel D) or vector backbone (panel F) probes (FIG.1). Confocal microscopy was conducted to reveal specific DNAhybridization, which generated the red dots (arrowed). The greenfluorescence (arrowhead) indicated BY2 nuclei stained with PicoGreen.Red fluorescence, green fluorescence and transmission images wereoverlapped for each panel.

FIG. 3. Visualization of plant nuclear DNA molecules and numbers ofT-DNA molecules inside plant cells. The BY2 cells were cocultivated withLBA4404(pIG121-Hm) for 1 d and subjected to the VOID procedure using theplant DNA [which was a 904-bp EcoRI fragment of an Arabidopsis thalianacDNA clone (corresponding to F16N3.18 of the genome sequence)] (panels Aand B) or T-DNA (panel C) probe (FIG. 1). Confocal microscopy wasconducted to reveal specific DNA hybridization, which generated the reddots (arrowed). The green fluorescence (arrowhead) indicated BY2 nucleistained with PicoGreen. Red and green fluorescence images wereoverlapped (panels A and C). To clearly reveal red dots, only redfluorescence image was shown in panel B. To count numbers of T-DNAmolecules inside BY2 cells, individual BY2 cells were reconstituted byoverlapping sequential 1-μm-laser-sections of confocal microscopy (panelC); the white bar denotes 10 μm.

FIG. 4. Time course of T-DNA trafficking inside plant cells. The BY2cells were cocultivated with LBA4404(pIG121-Hm) for 0 h (panel A), 2 h(panel B), 5 h (panel C), 1 d (panel D), 2 d (panel E), or 3 d (panelF). They were then subjected to the VOID procedure using the T-DNA probe(FIG. 1). Confocal microscopy was conducted to reveal specific DNAhybridization, which generated the red dots (arrowed). The greenfluorescence (arrowhead) indicated BY2 nuclei stained with PicoGreen.Red fluorescence, green fluorescence and transmission images wereoverlapped for each panel.

FIG. 5. Localization of VDI protein (SEQ ID NO:l) in tobacco BY2 andArabidopsis thaliana cells. Tobacco BY2 cells (A) and A. thaliana (B)cells were fixed and then subjected to the immunohistology usinganti-VDI as the primary antibody and anti-rabbit IgG-Cy3 as thesecondary antibody. Confocal microscopy was conducted to reveal thelocalization of VDI, which generated the red dots (arrowed). The greenfluorescence (arrowhead) indicated BY2 nuclei stained with PicoGreen.Red fluorescence, green fluorescence and transmission images wereoverlapped for each panel. There were not any significant signalsdetectable in the negative control (C) when preimmune serum instead ofanti-VDI was used. The VDI was located in the cytoplasm of cells andonly existed in some cells.

FIG. 6. GUS staining of tobacco BY2 cells. Tobacco BY2 cells werecocultivated with (A) or without (B) preinduced LBA4404(pIG121-Hm) for 3days and were then subjected to the GUS assay. Samples were then viewedunder light microscopy. The blue color spots (arrowhead) represented theGUS activity, which indicated that clusters of cells were transformed byA. tumefaciens. Only some plant cells were transformed by A. tumefaciensand gave blue color spots. Bar represents 126 μm.

FIG. 7. Coexistence of VDI and GUS protein in the same transformed BY2cells. Tobacco BY2 cells were cocultivated with preinducedLBA4404(pIG121-Hm) (A) or MX243(pIG121-Hm) (B) for 3 days. They werethen subjected to the double immunohistology assay. Confocal microscopywas conducted to reveal the location of VDI and GUS protein, whichgenerated the red dots (arrowed) and green dots (arrowhead),respectively. Red fluorescence, green fluorescence and transmissionimages were overlapped for each panel. Coexistence of VDI and GUS in thesame transformed BY2 cells indicated that only those cells producing VDIprotein could be transformed by A. tumefaciens.

FIG. 8. Colocalization of VDI protein and T-DNA molecules in the sametransformed BY2 cells. Tobacco BY2 cells were cocultivated withLBA4404(pIG121-Hm) (A) or MX243(pIG121-Hm) (B) for 1 day. They were thensubjected to VOID followed by immunohistology. Confocal microscopy wasconducted to reveal the location of VDI and T-DNA, which generated thered dots (arrowed) and green dots (arrowhead), respectively. Redfluorescence, green fluorescence and transmission images were overlappedfor each panel. Colocalization of VDI and T-DNA in the same transformedBY2 cells indicated that only those cells producing VDI protein couldreceive T-DNA delivered by Agrobacterium.

FIG. 9: Sequence homology between VirD2-Interacting protein (VDI; SEQ IDNO:l) and various members of the Sec3 family, including human Sec3 (SEQID NO:8), rat Sec3 (SEQ ID NO:9), mouse Sec3 (SEQ ID NO:10), and fruitfly Sec3 (SEQ ID NO:11).

DETAILED DESCRIPTION OF THE EMBODIMENTS

The invention is applicable to the monitoring of any exogenous nucleicacid in transit in a cell-containing biological sample.

The nucleic acid may be any DNA or RNA which has been introduced intothe cell. By “exogenous nucleic acid” is meant any nucleic acid which isnot already in the cell. The term encompasses nucleic acids such asplasmid constructs, viral nucleic acids, episomal DNA and cassettes,artificial chromosomes and naked DNA, which contain a sequence which isnot already present in the cell. It is contemplated that the inventionalso applies to the monitoring of nuclear fusion, where the DNA of onenucleus is distinguishable in sequence from the DNA of the othernucleus.

The exogenous nucleic acid is the target for in situ hybridization andmay be any introduced DNA target, or may be an introduced RNA such asthe genome of RNA viruses or RNA introduced by a retrovirus. The targetmay also be a nucleic acid which has been amplified by means such as thepolymerase chain reaction (PCR), so that additional copies of thenucleic acid targets are produced.

The term “biological sample” includes, but is not limited to, samples ofhuman, animal, microbial or plant origin such as human, animal,microbial or plant tissue sections, cell or tissue cultures, suspensionof human, animal or plant cells or isolated parts thereof, human oranimal biopsies, blood samples, cell-containing fluids and secretion.The introduced nucleic acids in whole cells contained in the biologicalsample are observed, not subcellular fractions.

Visualization of Introduced DNA (VOID)

The invention relates to the monitoring of nucleic acid in transit,after it has been introduced into the cell and as it progresses towardits endpoint. The nucleic acid is visualized while it is in flux, beforeor just as it reaches its endpoint. The endpoint is usually when theintroduced nucleic acid is in the nucleus and becomes integrated into orassociated with the host cell chromosomes, or engages the transcriptionmachinery for expression, or engages the cellular mechanism forself-replication. While it is understood that no living cell is actuallystatic at any point, by “in transit” is meant the progression of theintroduced nucleic acid towards its equilibrium or stable state at itsendpoint location in the cell. Usually this endpoint location is thenucleus of the cell into which the nucleic acid has been introduced.

The exogenous nucleic acid may be introduced into the cell by any meansknown in the art. The term “introduce” is sometimes used interchangeablyin the art with “transform” or “transfect”. Methods fortransforming/transfecting host cells with expression vectors arewell-known in the art and depend on the host system selected asdescribed in Ausubel et al., (Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons Inc., 1994). Means for introducingexogenous nucleic acids into cells include, but are not limited to,electroporation (see for example U.S. Pat. No. 5,273,525);liposome-mediated and lipoprotein mediated delivery (see for exampleU.S. Pat. No. 6,468,798); viral delivery systems such as retroviralvectors (see for example U.S. Pat. No. 6,410,313), adeno associatedviral vectors (see for example U.S. Pat. No. 6,153,436), herpes simplexviral vectors, and bacteriophage vectors (see for example U.S. Pat. No.6,054,312); cationic peptide mediated delivery (see for example U.S.Pat. 6,387,700); microneedle injection (see for example U.S. Pat. No.5,697,901); microparticle bombardment and Agrobacterium-mediated genedelivery, especially in plants (see for example U.S. Pat. No.5,932,782); and microinjection into the nucleus (see for example U.S.Pat. No. 6,498,285). The nucleic acid may be introduced first into thecytoplasm through the plasma membrane, or directly into the nucleusthrough the nuclear membrane. In a preferred embodiment, the nucleicacid is introduced into the cytoplasm through the plasma membrane.

Although the actual experiments described here relate toAgrobacterium-mediated transformation of tobacco cells, VOID should beapplicable to all eukaryotic cells, such as plant, yeast, fungal andanimal cells, including human cells, using any kind of nucleic deliverymethod known in the art and as described above.

In one embodiment, DNA is introduced into the target cells byAgrobacterium-mediated transformation. In nature, A. tumefaciens is asoil-borne bacterium that causes crown gall tumours on many plantspecies, particularly dicot plants. The bacterium transfers a specificsegment (T-DNA) of its tumour-inducing (Ti) plasmid into plant cells,where the T-DNA becomes integrated into the plant genome (Christie, P.J., 2001. Mol. Microbiol., 40(2), 294-305; Gelvin, S. B., 2000. Annu.Rev. Plant Physiol. Plant Mol. Biol., 51, 223-256; Zhu et al. 2000. J.Bacteriol., 182, 3885-3895; Zupan et al. 2000. Plant J., 23, 11-28). TheT-DNA contains the oncogenes that cause overproduction of plant hormonesand hence tumours. Therefore, Agrobacterium is a natural geneticengineer that transforms plants with its own genes for its own benefits.

The virulence (vir) genes located on the Ti plasmid are directlyresponsible for the T-DNA transfer process. The Agrobacterium genome hasbeen sequenced. Transformation using Agrobacterium has been modifiedsuch that useful genes can be introduced into many plant species withoutcausing tumours. Recently, it has been demonstrated that Agrobacteriumcan also transfer DNA into yeast, fungal and mammalian cells (P. Bundocket al. EMBO J. 14:3206 (1995); K. L. Piers et al. Proc. Natl. Acad. Sci.USA. 93:1613 (1996); M. J. de Groot et al. Nat. Biotechnol. 16:839(1998); T. Kunik et al. Proc. Natl. Acad. Sci. U.S.A. 98:1871 (2001)).This suggests that this system can be adopted for other eukaryoticcells.

The Agrobacterium system has several features that make it veryattractive as a general gene transfer vector. As the integration occursat fairly random positions, the T-DNA can be used as a tagging vector.The T-DNA can be also targeted to a specific site in the genome byhomologous recombination. Due to the accompanying VirD2 and VirE2proteins, the T-DNA is well preserved during its passage to the nucleus.The transformation is highly efficient due to the nuclear targeting bythe nuclear localization signals of VirD2 and VirE2. These nuclearlocalization signals are well conserved among eukaryotic cells.

VirD2 is an Agrobacterium virulence gene encoded protein that playsmultiple important roles in the transfer of T-DNA. First, VirD2 servesas an endonuclease that cleaves the bacterial T-DNA at the bordersequences. After cleavage, the VirD2 protein remains covalently attachedto the 5′ end of the T-strand. This would enable the VirD2 protein toserve as a pilot protein that guides the passage of the T-strand fromAgrobacterium into plant cells.

There are a number of plant proteins that specifically interact withVirD2. These proteins are well conserved in the eukaryotic cells,including plant, yeast, fungal and animals cells. Since T-DNA can bedelivered by the bacterium into plant, yeast, fungal and mammalian cellnuclei, this suggests that the DNA trafficking pathway is well conservedamong eukaryotic cells.

The structure of the integrated T-DNA is similar regardless of the hostgenome wherein the integration took place. This indicates that the samemolecular mechanism of T-DNA integration is used by different eukaryoticspecies. Recently the proteins that mediate non-homologous T-DNAintegration have been identified using the yeast Saccharomycescerevisiae as a model. These included the yeast Ku70-Ku80 heterodimer,DNA ligase IV and the Mrell, Xrs2, Rad50 complex. These proteins are allknown to be involved in double strand break (DSB) repair bynon-homologous end-joining. As these proteins are conserved in othereukaryotes including animals and plants, this suggests that the samemechanism of non-homologous T-DNA integration is used by all speciesstudied so far. This demonstrates that Agrobacterium delivery of T-DNAoffers an feasible system to study DNA trafficking and DSB repair ineukaryotic cells.

Sample Preparation

At various intervals after the nucleic acid is introduced into the cell,the cell is treated in preparation for in situ hybridization. Theappropriate treatment will depend on the type of sample to be examined,as known in the art. During the treatment, the sample will be subject tofixation and, if required, permeabilization.

In one aspect of the invention, the sample is deposited onto a solidsupport. The particular techniques appropriate for depositing the sampledepends on the type of sample. Such techniques include, for example,sectioning of tissue as well as smearing or cytocentrifugation of cellsuspensions.

The types of solid supports are known in the art. Supports which may beutilized include, but are not limited to, microporous beads or sponges,glass, Scotch tape (3M), nylon, Gene Screen Plus (New England Nuclear),magnetic particles and nitrocellulose. Most preferably glass microscopeslides are used. The use of these supports and the procedures fordepositing specimens thereon are known in the art. The choice of supportmaterial will depend upon need for the procedure used to visualize oranalyze cells and the quantitation procedure used. Some filter materialsare not uniformly thick and, thus, shrinking and swelling during in situhybridization procedures is not uniform. In addition, some supportswhich autofluoresce will interfere with the determination of low levelfluorescence. Glass microscope slides are most preferable as a solidsupport since they have high signal-to-noise ratios and can be treatedto better retain tissue.

Prior to hybridization, the sample is suitably treated with variouschemicals to facilitate the subsequent reactions. The actualpretreatment will depend on the type of sample to be analysed and onwhether DNA or RNA sequences are to be detected. For monitoring RNA, thesample may need to be treated as soon as possible after the RNA isintroduced into the cell. It may be advantageous to treat the samplewith DNase to minimise the background noise when the target sequence isRNA. By fixing the cells in the sample, the morphological integrity ofthe cellular matrix and of the nucleic acids within the cell ispreserved.

Fixing may be by means of chemical fixation or freezing. When freezingis used for preservation of a sample, the sample may be frozen in liquidnitrogen and stored at −80° C. Prior to the analysis of the nucleicacid, the frozen sample is cut into thin sections and transferred toe.g. pre-treated slides. This can e.g. be carried out at a temperatureof −20° C. in a cryostat. The tissue sections may suitably be stored at−80° C. until use.

In preparation for hybridization, the biological sample may be treatedwith a fixative, including a precipitating fixative such as acetone.Alternatively, the biological sample is incubated for a short period ina solution of buffered formaldehyde. The biological sample can also betransferred to a fixative such as buffered formaldehyde for 12 to 24hours. Following fixation, the biological sample may be embedded inparaffin forming a block from which thin sections can be cut.

Fixatives are compounds that kill a cell but preserve its morphologyand/or nucleic acids for an extended period of time. They act either bycreating covalent linkages between cellular molecules or byprecipitating certain intracellular molecules. Cross-linking fixativesinclude formaldehyde, glutaraldehyde, paraformaldehyde,ethyldimethyl-aminopropyl-carbodiimide, and dimethylsilserimidate.Precipitants include ethanol, acetic acid, methanol, acetone, andcombinations thereof. It is further preferred that glacial acetic acidbe included as a fixative when the cells are to be monitored by flowcytometry. If a cross-linking fixative is used, paraformaldehyde (0.1%v/v to 4% v/v is preferred, 0.5% v/v to 1% v/v is especially preferred;2 hours to 20 hours preferred). Formaldehyde and gluteraldehyde areamong the other possibilities. Fixatives are used at concentrationswhich do not destroy the ability of the cell's nucleic acids tohybridize to the probe. Fixatives and hybridization of fixed cells, ingeneral, are discussed in WO 90/02173 and WO 90/02204. See also U.S.Pat. No. 5,719,023, U.S. Pat. No. 5,888,733 and U.S. Pat. No. 5,521,061for general discussions of in vitro hybridization.

Prior to hybridization, the biological sample may be de-waxed andrehydrated using standard procedures.

For all sample preparation, the nucleic acids are fixed in morphologicalrelationship with cellular structure allowing hybridization to becarried out in situ. The nucleic acids are not extracted from thecellular material and hybridization is not carried out in solution.

If RNA sequences are the target for hybridization, degradation byribonucleases during the prehybridization steps should be avoided. Allequipment and solutions used for pretreatment as well as forhybridization should be appropriately treated to remove nucleases. Suchinactivation techniques are well known in the literature and may beperformed according to standard procedures.

In preparing a biological sample for in situ hybridization, it may benecessary to treat the sample so as to permeabilize the material andpreserve the morphology.

Permeabilization may be necessary in order to ensure sufficientaccessibility of the probe to the target nucleic acid sequences. Thetype of treatment will depend on several factors, for instance on thefixative used, the extent of fixation, the type and size of sample usedand the length of the probe. The treatment may involve exposure toprotease such as proteinase K, pronase or pepsin, diluted acids,detergents or alcohols or a heat treatment.

For biological samples such as plant where the cells have cell walls, itmay be necessary to remove the cell wall to allow the probe access tothe target nucleic acid. The cell wall may be removed by digestion witha cell wall-digesting enzyme such as cellulase. In one embodiment, thecell wall is removed after the cells have been fixed, but beforepermeabilization.

Permeabilizing agents include, but are not limited to, detergents suchas Brij 35, Brij 58, sodium dodecyl sulfate, CHAPS, and TRITON X-100.Depending on the location of the target nucleic acid, the permeabilizingagent is chosen to facilitate probe entry through the cell membranes,preferably the plasma membrane. For instance, 0.05% Brij 35 or 0.1%TRITON X-100 will permit probe entry through the plasma membrane but notthe nuclear membrane. Alternatively, sodium deoxycholate will allowprobes to traverse the nuclear membrane. Thus, in order to restricthybridization to the cytoplasmic structures, nuclear membranepermeabilizing agents are avoided. Such selective subcellularlocalization may improve the specificity and sensitivity of detection byminimizing probe hybridization to complementary nuclear sequences whenthe target sequence is located in the cytoplasm.

In situ Hybridization

In situ hybridization may be performed using any of the methods known inthe art (see Jong et al 1999. High resolution FISH in plants—techniquesand applications. Trends in Plant Science, 4, 258-263 and Nath J andJohnson KL 2000. A review of fluorescence in situ hybridization (FISH):Current status and future prospects. Biotech Histochem 75: 54-78). Thebasic steps involve hybridization of a probe to the exogenous nucleicacid, washing the sample to remove non-specific binding, and visualizingthe bound probe.

A probe is defined as genetic material DNA, RNA, or oligonucleotides orpolynucleotides comprised of DNA or RNA. The DNA or RNA may be composedof the bases adenosine, uridine, thymidine, guanine, cytosine, or anynatural or artificial chemical derivatives thereof. The probe is capableof binding to a complementary or mirror image target nucleic acidthrough one or more types of chemical bonds, usually through hydrogenbond formation. The extent of binding is referred to as the amount ofmismatch allowed in the binding or hybridization process; the extent ofbinding of the probe to the target sequences also relates to the degreeof complementarity to the target sequences. The size of the probe isadjusted to be of such size that it forms stable hybrids at the desiredlevel of mismatch; typically, to detect a single base mismatch requiresa probe of approximately 12-50 bases. Larger probes (from 50 bases up totens of thousands of bases) are more often used when the level ofmismatch is measured in terms of overall percentage of similarity of theprobe to the target cellular genetic sequence. The size of the probe mayalso be varied to allow or prevent the probe from entering or binding tovarious regions of the genetic material or of the cell. Similarly, thetype of the probe (for example, using RNA versus DNA) may accomplishthese objectives. The size of the probe also affects the rate of probediffusion, probability of finding a cellular target match, etc.

The length of a probe affects its diffusion rate, the rate of hybridformation, and the stability of hybrids. As a general guide, to detecttarget RNA, small probes (50-150 bases) may allow the most sensitive,rapid and stable signal. A mixture of short probes (50-150 bases) areprepared which span the entire length of the target sequence. Forexample, if the target sequence were 1000 bases long, about 10-20“different” probes of 50-100 bases would be used in the hybrid solutionto completely cover all regions of the target sequence. To detect targetDNA, smaller probes (15-50 bases) may be utilized.

The concentration of the probe affects several parameters of the in situhybridization reaction. High concentrations are used to increasediffusion, to reduce the time of the hybridization reaction, and tosaturate the available cellular sequences. In an embodiment, probeconcentrations of 1-10 μg/ml or 2.5 μg/ml are used.

Nucleic acid probes can be prepared by a variety of methods known in theart. The probe may be constructed or obtained by one of a number ofstandard methods. Many probes, such as various satellite DNA sequencesare commercially available in single-stranded or double-stranded form.Other probes can be obtained either directly from viruses, plasmids andcosmids or other vectors carrying specific sequences, or, if desired, byrestriction digest of the source of the probe DNA, such as a vector,followed by electrophoretic isolation of specific restriction digestionfragments. Probes obtained in this manner are typically indouble-stranded form, but may, if required, be subcloned insingle-stranded vectors, such as an M13 phage vector.

Purified double-stranded sequences of DNA (dsDNA) can be labeled intactby the process of nick translation or random primer extension. Theability of double-stranded probes to hybridize to nucleic acidsimmobilized within cells is compromised by the ability of thecomplementary strands to hybridize to each other in solution prior tohybridization with the cellular nucleic acids. Single-stranded DNA(ssDNA) probes do not suffer this limitation and may be produced by thesynthesis of oligonucleotides, by the use of the single-stranded phageM13 or plasmid derivatives of this phage, or by reverse transcription ofa purified RNA template. The use of single-stranded RNA (ssRNA) probesin hybridization reactions potentially provides greater signal-to-noiseratios than the use of either double or single-stranded DNA probes.Regardless of whether a dsDNA, a ssDNA, or a ssRNA probe is used in thehybridization reaction, there must be some means of detecting hybridformation. The means of detecting hybrid formation utilizes a probe“labeled” with some type of detectable label.

For a discussion of probes, see Handbook of Fluorescent Probes andResearch Products, Ninth Edition by Richard P. Haugland (2002) MolecularProbes.

The probe is labeled with a reporter or ligand or moiety which allowsdetection of the targeted sequence in situ. The probes may be detectablylabeled prior to addition to the hybridization solution. Alternatively,a detectable label may be selected which binds to the reaction product.Probes may be labeled with any detectable group for use in practicingthe invention. Such detectable group can be any material having adetectable physical or chemical property. Such detectable labels havebeen well-developed in the field of immunoassays and in general most anylabel useful in such methods can be applied to the present invention.Particularly useful are enzymatically active groups, such as enzymes(see Clin. Chem., 22:1243 (1976)), enzyme substrates (see British Pat.Spec. 1,548,741), coenzymes (see U.S. Pat. Nos. 4,230,797 and 4,238,565)and enzyme inhibitors (see U.S. Pat. No. 4,134,792); fluorescers (seeClin. Chem., 25:353 (1979); chromophores; luminescers such aschemiluminescers and bioluminescers (see Clin. Chem., 25:512 (1979));specifically bindable ligands; proximal interacting pairs; andradioisotopes such as ³H, ³⁵S, ³²P, ¹²⁵I and ¹⁴C. For autoradiographicdetection, the reporter is a radiolabel, such as ³²P-labeled probeformed, for example by nick translation or polymerase chain reaction inthe presence of labeled nucleotides.

For fluorescence detection, the probe may be labeled with one of aselection of fluorescence groups, such as FITC, BODIPY, Texas Red, orCascade Blue which is excitable in a specific wavelength, such as 490,540, and 361 nm. The groups are derivatized to 3′ or 5′ probe ends or byincorporation or reaction at internal positions, according to standardmethods.

Alternatively, the probes may be labeled with a ligand-type reportersuch as biotin, digoxigenin, or bromodeoxyuridine or other modifiedbases including fluorescein-11-dUTP. The probe reporter groups aredetected, in situ, by reaction of the hybridized probe with a secondaryreporter molecule which (a) binds specifically and with high affinity tothe probe ligands, and (b) contains a detectable reporter. The bindingmoiety of the secondary molecule may be avidin or streptavidin, forbinding to biotinylated nucleotides, anti-digoxigenin antibody, forbinding to digoxigenin-labeled nucleotides, and anti-BrdUrd antibody forbinding to BrdUrd-labeled probe.

The detectable reporter in the secondary molecule is typically afluorescence label, but may also be a radiolabel, for autoradiographicdetection, an antibody, an enzyme, for colorimeteric orchemiluminescence detection in the presence of a suitable substrate, orcolloidal gold for use in electron microscopic visualization.

Nucleic acid hybridization is a process well known in the art where twoor more mirror images or opposite strands of DNA, RNA, oligonucleotides,polynucleotides, or any combination thereof recognize one another andbind together through the formation of some form of either spontaneousor induced chemical bond, usually a hydrogen bond. The degree of bindingcan be controlled based on the types of nucleic acids coming together,and the extent of “correct” binding as defined by normal nucleic acidscoming together, and the extent of “correct” binding as defined bynormal chemical rules of bonding and pairing.

Hybridization of the probe to the target introduced nucleic acid can beperformed using the fixed and permeabilized preparations prepared asdescribed above. If double-stranded target such as chromosomal or DNAsequences are to be detected, a treatment to separate the two strandsmay be necessary. This separation of the strands can be achieved byheating the sample in the presence of the hybridization mixture to atemperature sufficiently high and for a time period sufficiently long todissociate the strands. Typically, heating at a temperature of 90° C. to95° C. for a period of 5 to 15 minutes is suitable.

The hybridization buffer may contain a hybrid destabilizing agent in anamount effective to decrease the melting temperature of hybrids formedbetween the nucleic acid to be determined and the binding partner so asto increase the ratio between specific binding and non-specific binding(see U.S. Pat. No. 5,888,733). This agent allows the hybridization totake place at a lower temperature than without the agent. In traditionalnucleic acid hybridization, such agent is called a denaturing agent.Hybridization and denaturing may be carried out simultaneously using asuitable amount a hybrid destabilizing agent in combination with asuitable temperature for the treatment. Examples of hybrid destabilizingagents are formamide, ethylene glycol and glycerol and these agents maypreferably be used in a concentration above 10% and less than 70%. Theconcentration of formamide may more preferably be from 20% to 60%, mostpreferably from 30% to 50%. The concentration of ethylene glycol maymore preferably be from 30% to 65%, most preferably 65%. Theconcentration of glycerol may more preferably be from 45% to 60%, mostpreferably 50%.

It is often advantageous to include macromolecules or polymers such asdextran sulphate, polyvinylpyrrolidone and/or ficoll. In the presence ofsuch macromolecules or polymers, the effective concentration of theprobe at the target is assumed to be increased. Dextran sulphate may beadded in a concentration of up to 15%. Concentrations of dextransulphate of from 2.5% to 12.5% may be advantageous.

Other important hybridization parameters are temperature, concentrationof the probe and hybridization time. A skilled person will readilyrecognize that optimal conditions for various starting materials willhave to be determined for each of the above-mentioned parameters.

Following hybridization, the prepared biological sample is washed toremove any unbound and any non-specifically bound probes. During thepost-hybridization step, appropriate stringency conditions should beused in order to remove any non-specifically bound probe. Stringencyrefers to the degree to which reaction conditions favour thedissociation of the formed hybrids and may be enhanced, for instance byincreasing the washing temperature and incubation time. Saltconcentration is often used as an additional factor for regulating thestringency. Examples of useful buffer systems are Tris-Buffered-Saline(TBS), standard citrate buffer (SSC) or phosphate buffers. A convenientTBS buffer is 0.05M Tris/HCl, 0.15M NaCl, pH 7.6. The SSC buffercomprises 0.15M sodium chloride and 0.015M trisodium citrate, pH 7.0.Typically, washing times from 25 to 30 minutes may be suitable. Washingperiods of two times 10 minutes or 3 times 5 minutes in a suitablebuffer may also be suitable.

Where the preparation is deposited onto slides, the hybridizationresults may be visualized using well known immunohistochemical stainingmethods to detect the labelling on the probe. When fluorescent labelledprobes are used, the hybrids may be detected using an antibody againstthe fluorescent label which antibody may be conjugated with an enzyme.The fluorescent label may alternatively be detected directly using afluorescence microscope, or the results may be automatically analysed ona fluorescent-based image analysis system. The signal may be visualizedby confocal microscopy, fluorescence microscopy, or electron microscopy.

When biotin labelled binding partners are used, the hybrids may bedetected using an antibody against the biotin label which antibody maybe conjugated with an enzyme. If necessary, an enhancement of the signalcan be generated using commercially available amplification systems suchas the catalyzed signal amplification system for biotinylated probes(DAKO K 1500).

Applications of VOID

VOID is useful for monitoring introduced nucleic acids whenever thenucleic acid is in transit. As examples, a number of specificapplications are contemplated below.

Applications of VOID utilize VOID's ability to reveal the number andlocation of the introduced nucleic acid, as well as its ability toidentify the cells that are competent for receiving nucleic acidmolecules. In addition, VOID can be used to determine the fate andtiming of nucleic acid being delivered into the cells; that is, VOID canbe used to identify where the nucleic acid molecules have gone after thenucleic acid has entered the cell and where they are in the cell at anytime after cell entry. By the use of VOID, drawbacks associated withcurrent techniques for monitoring nucleic acid delivery andtransformation may be avoided.

VOID can be used to help the development of genetically modifiedproducts, including gene therapy vectors, gene therapy delivery systems,as well as assess the safety of gene therapy treatments. VOID can alsobe used to facilitate development of DNA vaccines, DNA vaccine vectors,DNA vaccine delivery systems, and safety assessment of DNA vaccines. Inaddition, VOID can be used in the development of transgenic or geneticengineered products such as genetically modified food crops.

The use of VOID in genetic modification is significant because fordevelopment of safe and effective genetically modified products or genedelivery vectors such as those used in gene therapy, one needs to knowthe number, location and fate of the nucleic acid delivered into thetargeted cells. Because VOID can reveal the number and location of thenucleic acid delivered into the cells and identify the cell typescompetent for receiving the nucleic acid, this facilitatesidentification and development of DNA delivery systems and formulations,and generate efficient nucleic acid delivery where the desired number ofnucleic acid molecules are delivered into the desired target cells. Thususe of VOID can considerably speed up the development of geneticallymodified products including gene therapy products and DNA vaccines.

The use of VOID in the development of transgenic or genetic engineeredproducts is also significant because marker or reporter genes are nolonger necessary to monitor nucleic acid delivery and transformation. Asa result, transgenic products may be produced that are free of marker orreporter genes which may be of concern for the environment and humanhealth.

VOID can also be used to determine the cell types that can efficientlyreceive the nucleic acid intended for transformation, thus identifyingthe best cells and tissues or organs for nucleic acid uptake. Asillustrated below with the cellular protein VirD2-Interacting protein(VDI; SEQ ID NO:1), VOID is also useful for identifying molecularmarkers associated with a cell's competence to receive DNA molecules.

I. Assessing Transformation Status and Efficiency

In one embodiment, VOID is used to monitor T-DNA introduced into plantcells by A. tumefaciens.

Agrobacterium tumefaciens is a natural genetic engineer that transformsplants with its own genes for its own benefits (P. J. Christie. Mol.Microbiol, 40:294 (2001); S. B. Gelvin. Annu. Rev. Plant Physiol. PlantMol. Biol. 51:223 (2000); J. Zhu et al. J. Bacteriol. 182:3885 (2000);J. Zupan et al. Plant J. 23:11 (2000)). The bacterium transfers aspecific segment (T-DNA) of its tumor-inducing (Ti) plasmid into plantcells, where the T-DNA becomes integrated into the plant genome. Thissystem has been used as the workhorse to introduce various genes intomany different plant species.

Recently, it has been demonstrated that Agrobacterium can also transferT-DNA into yeast (Bundock et al. 1995. EMBO J. 14:3206-14; Piers et al.1996. Proc Natl Acad Sci USA 93:1613-8), fungal (de Groot M J et al.1998. Nat Biotechnol. 16:839-42) and human cells (Kunik et al. 2001.Proc Natl Acad Sci USA. 98:1871-1876). Major bacterial genes involved inthe gene transfer process have been identified and characterized(Christie, P. J., 2001. Mol. Microbiol., 40(2), 294-305; Gelvin, S. B.,2000. Annu. Rev. Plant Physiol. Plant Mol. Biol., 51, 223-256; Zhu etal. 2000. J. Bacteriol., 182, 3885-3895; Zupan et al. 2000. Plant J.,23, 11-28).

A. tumefaciens genome has been sequenced (Wood, D. W., et al 2001.Science, 294, 2317-2323; Goodner, B., et al 2001. Science, 294,2323-2328). The Ti plasmid harbored virulence (vir) genes are directlyresponsible for the processing and transfer of T-DNA; all of them areinduced by plant signal molecules, such as acetosyringone (AS). VirD2generates a linear single-stranded (ss) DNA molecule (the T-strand) bynicking the border repeats that delineate the T-DNA (Stachel, S. E.,Nester, E. W., 1986. EMBO J., 5, 1445-1454; Yanofsky G, et al. Cell.Nov. 7, 1986;47(3):471-7). The T-strand is transferred into plant nucleipresumably in the form of nucleoprotein complex (T-complex)(Ziemienowicz et al. 2001. The Plant Cell, 13, 369-384) consisting ofone VirD2 molecule, one T-strand and many VirE2 molecules. The virB geneproducts and VirD4 form a membrane structure responsible fortransferring the T-DNA (Fullner et al. 1996. 273, 1107-1109) in aprocess mechanistically analogous to conjugation (Christie, P. J., 2001.Mol. Microbiol., 40(2), 294-305) and promiscuous enough to be applicableto exceptionally diverse recipients like plant, yeast, fungal and humancells.

Plant proteins that interact with VirD2 and VirE2 have been identified(Ballas, N., Citovsky, V., 1997. Nuclear localization signal bindingprotein from Arabidopsis mediates nuclear import of Agrobacterium VirD2protein. Proc. Natl. Acad. Sci. USA, 94, 10723-10728; Deng et al. 1998.Agrobacterium VirD2 protein interacts with plant host cyclophilins.Proc. Natl. Acad. Sci. USA, 95, 7040-7045; Tzfira et al. 2001. VIP1, anArabidopsis protein that interacts with Agrobacterium VirE2, is involvedin VirE2 nuclear import and Agrobacterium infectivity, EMBO J., 20,3596-3607); some plant proteins have been implicated in the nuclearimport of T-complex or T-DNA integration (Gelvin, S. B. 2000. Annu. Rev.Plant Physiol. Plant Mol. Biol., 51, 223-256). However, individual T-DNAmolecules have never been visualized inside eukaryotic cells during theT-DNA passage in vivo.

It is still unknown how many T-DNA molecules can be delivered into asingle eukaryotic cell and how many of them can be transferred fromcytoplasm into nucleus. The mode of T-DNA passage into and througheukaryotic cytoplasm remains elusive. It is not well established whysome eukaryotes including certain monocotyledonous plants arerecalcitrant to Agrobacterium-mediated transformation, while manyreceptive eukaryotes like dicotyledonous plants can be efficientlytransformed, with a variable number of integrated T-DNA copies. To helpaddress these questions, VOID was used to monitor T-DNA moleculestrafficking inside plant cells. This procedure may be used not only todissect the T-DNA trafficking pathway(s) inside eukaryotic cells, butalso to monitor the T-DNA transfer and passage inside eukaryotic cells.

Part of a T-DNA fragment was labeled with digoxigenin-11-dUTP (DIG); theT-DNA probe was allowed to permeate the plant cells that had beencocultivated with A. tumefaciens cells harboring a vector plasmid(pIG121-Hm) containing the T-DNA (FIG. 1). The T-DNA molecules deliveredinto the plant cells were allowed to hybridize with the probe DNAlabeled with DIG, which could be bound to anti-DIG antibody conjugatedwith rhodamine which gave red fluorescence when excited by 543 nm light.As shown in FIG. 2A, many red dots could indeed be detected underconfocal microscope when the tobacco BY2 cells were allowed tococultivate with LBA4404(pIG121-Hm). The nuclei were counterstained withPicoGreen, which could give green fluorescence, in order to gauge thesubcellular locations of T-DNA molecules.

To verify that the red dots indeed corresponded to the T-DNA insideplant cells rather than T-DNA from contaminating A. tumefaciens, thefollowing experiments have been conducted. First, the bacteria wereextensively washed away after the cocultivation; indeed very fewbacteria could be found after the cocultivation and then the VOIDprocedure. Second, it was determined if the red dots were specificallycorrelated with the bacterial ability to deliver the T-DNA. The sameplasmid pIG121-Hm was introduced into a virb mutant MX243 (Stachel &Nester, E. W., 1986. EMBO J., 5, 1445-1454; P. J. Christie. Mol.Microbiol. 2001. 40:294), which is unable to deliver the T-DNA, and avirD2 mutant WR1715 (Stachel & Nester, E. W., 1986. EMBO J., 5,1445-1454; Shurvinton et al. 1992. Proc. Natl. Acad. Sci. USA, 89,11837-11841; Zupan et al. 2000. Plant J., 23, 11-28), which is unable toproduce the T-DNA. When the cocultivation and VOID were conducted withthese mutants, no T-DNA signal was detected inside plant cells (FIG.2B&C). This indicated that the signal was due to the hybridization ofT-DNA with the specific probe.

Finally, it was determined if the signal was due to the untransferredT-DNA still residing inside A. tumefaciens cells. The A. tumefacienschromosomal gene aopB (Jia Y. H., L. P. Li, Q. M. Hou and S. Q. Pan.2002. An Agrobacterium gene involved in tumorigenesis encodes an outermembrane protein exposed on the bacterial cell surface. Gene.284:113-124) was labelled and the same VOID procedure was carried out.As shown in FIG. 2D, no signal was detectable in the samples when theaopb probe was used for hybridization. This indicated that the VOIDprocedure could not detect any DNA residing inside the bacterial cells,presumably because of insufficient permeabilization of the bacterialmembranes. These confirmed that the VOID procedure could detect theT-DNA delivered into plant cells.

II. Numbers and Locations of DNA Molecules Delivered into Cells

Subsequently, it was important to know if the VOID procedure couldreveal the location and number of individual T-DNA molecules. To dothis, an Arabidopsis thaliana cDNA (corresponding to F16N3.18 of thegenome sequence) clone was chosen that could hybridize to the BY2genomic DNA as demonstrated by Southern analysis. When this cDNAfragment was used to probe BY2 cells, VOID consistently revealed 1 or 2red dots in each single cell nucleus (FIG. 3A&B). No hybridizationsignal was detected in the cytoplasm of BY2 cells, suggesting that VOIDcould detect DNA and not RNA molecules as RNase A was added.Conceivably, 2 red dots could been seen if the confocal section in focuscontained two nuclear DNA molecules that could specifically hybridizewith the cDNA fragment; 1 red dot would be seen if only one was infocus. This indicated that VOID could reveal the locations and numbersof individual T-DNA molecules present throughout the BY2 cells.

When a virE2 mutant MX358 (Stachel & Nester. 1986. EMBO J., 5,1445-1454; Winans et al. 1987. Nucleic Acid Research 15: 825-836; Zupanet al. 2000. Plant J., 23, 11-28) was used to conduct the cocultivationand VOID procedure, very few T-DNA molecules were found inside the BY2cells (FIG. 2E). This suggests that mutation at virE2 may severelyattenuate the bacterial ability to deliver the T-DNA into plant cells,which is consistent with the recent evidence that the VirE2 protein canform a membrane channel to facilitate ssDNA transport (Dumas et al.2001. Proc. Natl. Acad. Sci. USA, 98, 485-490). It may also indicatethat T-DNA molecules were quickly degraded inside plant cells in theabsence of the VirE2 protein, which is known to bind to T-DNA andpresumably can protect T-DNA from degradation (Zhu et al. 2000. J.Bacteriol., 182, 3885-3895; Zupan et al. 2000. Plant J., 23, 11-28).

To determine whether DNA segments outside the T-DNA could also betransferred into the plant cells, a fragment of pIG121-Hm vectorbackbone outside the left border (FIG. 1) was used to probe the BY2cells cocultivated with LBA4404(pIG121-Hm). As shown in FIG. 2F, thevector backbone fragment outside the T-DNA were detected in the plantcells. However, the numbers of such molecules appeared to be about 18times lower as compared to the T-DNA molecules. This suggests that thebinary vector backbone can be transferred into the plant cells alongwith the T-DNA, although the frequency of such an event is lower thanthat for the T-DNA alone. In fact, previous experiments demonstratedthat A. tumefaciens could transfer DNA sequences outside the T-regionand even plasmids into plant cells (Gardner and Knauf. Science, Vol.231, No. 4739. Feb. 14, 1986, pp. 725-727; Buchanan-Wollaston et al.1987. Nature 328: 172-175). These further demonstrated that the VOIDprocedure could reliably reveal data consistent with the previousobservations.

The numbers of T-DNA molecules inside BY2 cells were counted afterreconstitution of individual plant cells from sequential1-μm-laser-sections of confocal microscopy. Roughly half of the BY2cells did not contain any T-DNA molecules, suggesting that thecompetency of BY2 cells was important for BY2 cells to receive the T-DNAdelivered by A. tumefaciens. The numbers of T-DNA molecules inside theBY2 cells that had received T-DNA also varied greatly. Some receivedhundreds of T-DNA molecules (FIG. 2A); some had only a few (FIG. 3C). Ina typical cocultivation experiment, the average numbers of T-DNAmolecules inside the cytoplasm of a single BY2 cell that received T-DNAwere around 63.

III. Time-Course of DNA Delivery into Cells

BY2 cells were cocultivated with LBA4404(pIG121-Hm) for different timeintervals. As shown in FIG. 4B, the T-DNA was barely detectable in theplant cytoplasm at 2 h of cocultivation, which is consistent with theprevious observations that T-DNA could be detected at 2 h afterinfection of plants with A. tumefaciens (Virts & Gelvin. 1985. J.Bacteriol., 162, 1030-8; Narasimhulu et al. 1996. Plant Cell 8:873-886). Most of the T-DNA molecules entered the plant cytoplasm in 5 h(FIG. 4C). At 1 day of cocultivation, all the T-DNA molecules stillremained in the cytoplasm of the BY2 cells (FIG. 4D). At 2 days ofcocultivation, all the T-DNA molecules were inside the plant nuclei(FIG. 4E). This is consistent with the β-glucuronidase (GUS) stainingexperiments, which could indicate the expression of T-DNA. It was foundthat the GUS activity was not detectable until 2 days of cocultivation(data not shown). These suggest that the T-DNA could enter plant cellsvery fast (about 5 h), but it took a longer period (about 2 d) for T-DNAto enter plant nuclei. The T-DNA harbored genes were quickly expressedupon entry of T-DNA into the nuclei.

IV. Eliminating need for Selection/Marker to Identify Transformant

Widespread use and the subsequent spreading of specific marker genes ingenetically modified products has raised concerns about the safety andenvironmental effects of these products. Currently genetically modifiedcells are selected on the basis of expression of a functional product ofthe DNA delivered; the functional product may be the desired product, ormore commonly, the product of a gene that is introduced into the cellalong with the gene encoding the desired product. These latter productsare commonly referred to as selection markers or reporters.

Selection markers are those genes which, upon expression, produces aprotein capable of facilitating the isolation of cells expressing themarker. Examples of markers include neomycin, hypoxanthine phosphribosyltransferase, puromycin, dihydrooratase, glutamine synthetase, histidineD, carbamyl phosphate synthase, dihydrofolate reductase, multidrugresistance I, aspartate transcarbamylase, xanthine-guaninephosphoribosyl transferase, or adenosine deaminase. In plants, markersare used that confer on the transformed plant cells resistance to abiocide or an antibiotic, such as kanamycin, G 418, bleomycin,hygromycin, or chloramphenicol, etc.

Reporter genes encode a functional product such that when the gene isexpressed, the product is detectable by means of a suitable assay.Common reporter genes include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase or Green Fluorescent Protein (GFP) gene.

Since the function of the selection marker or reporter is to permitidentification and selection of the transformed cell, the marker orreporter becomes unnecessary after the transformant is identified. Thusideally the transformed cell would contain only the desired nucleicacid, with as little as possible of non-essential material, such asmarker genes and remnants of the DNA used for cloning.

In one embodiment, VOID is used to identify cells that contain the T-DNAintroduced into plant cells by A. tumefaciens. Identification oftransformed cells is determined by monitoring entry of the T-DNA intothe nucleus. As shown in FIG. 4C, most of the T-DNA molecules enteredthe cytoplasm in 5 h. However, none of them entered the nucleus even at1 day of cocultivation (FIG. 4D). Once they entered the nucleus in 2days, no T-DNA molecule could be found in the cytoplasm (FIG. 4E). Theseresults indicate that the T-DNA molecules appeared to have moved intothe nucleus together in one wave. The data also suggest that the T-DNAtrafficking inside plant cells is not a simple diffusion process. If itwas a diffusion process, some T-DNA molecules would have arrived insidethe nuclei at 1 day of cocultivation, since many T-DNA molecules werealready present throughout the cytoplasm at 5 h of co-cultivation. Inaddition, some of them would have remained inside the cytoplasm whileothers entered the nucleus.

Previous experiments with an in vitro nuclear import system demonstratedthat T-complex could be translocated from plant cytoplasm into nucleusvery quickly (within 20 min) (Ziemienowicz et al. 2001. The Plant Cell,13, 369-384). The in vivo data indicated that T-DNA trafficking insideplant cytoplasm was very slow. It appeared that the T-DNA moleculesinside cytoplasm were somehow unavailable for or prohibited from thequick nuclear import process. Consistent with this, T-DNA traffickinginside plant cytoplasm appeared to occur in a coordinated manner, asonly one wave of T-DNA trafficking was apparent during the 3-daycocultivation (FIG. 4).

Thus by using VOID, the cycle of T-DNA import into the nucleus isobserved, and cells which have undergone T-DNA import can be identifiedwithout having to use a selection or marker product.

V. Assessing Risk Associated with Transformation or Nucleic AcidDelivery

It is important to know how many DNA molecules have been delivered intothe cells and where the molecules have gone. This is because an excessnumber of DNA molecules delivered into the cells could potentially leadto unwanted integration of some of the DNA molecules into vital sites ofthe genome, which could lead to serious health problems to the host.Since VOID can be used to determine the number and location of thenucleic acid delivered into the cells, VOID can be used to calculate therisk associated with a genetically modified product transformed using aparticular gene delivery process.

By monitoring the fate of the exogenous nucleic acid in the cell, onecan assess the relative safety not only of a particular gene deliverytechnique, but also the risk associated with using a particular nucleicacid delivery vehicle. For example, the uptake, number, location, andmovement of a desired gene may be monitored when the gene is deliveredas part of a naked plasmid, as part of a liposomal complex, as part of aviral delivery system such as retroviral vectors, adeno associated viralvectors, herpes simplex viral vectors, and bacteriophage vectors; aspart of a nucleic acid associated with cationic peptides, or as part ofan Agrobacterium tumefaciens vector. Depending on the context in whichthe desired gene is delivered, the desired gene may be stable orunstable in the cell; the gene may also be capable of replicating atdifferent rates and efficiency. All these factors are important inassessing the desirability of using a particular gene delivery system.

In one embodiment, VOID is used to assess the risk associated with A.tumefaciens-mediated transformation of plant cells. The efficiency ofT-DNA molecules that moved from cytoplasm into nucleus was assessed bydetermining the ratio of the numbers of T-DNA molecules inside cytoplasmto those inside nucleus. In a typical cocultivation, it was found that 1in 6 T-DNA molecules moved from cytoplasm to nucleus (FIG. 4D&E). Thisdemonstrated that not every T-DNA molecule delivered into plantcytoplasm could make all the way into the nucleus, suggesting that thetransformation process involved a shot-gun approach. Since the VOIDprocedure could reveal the number and location of T-DNA moleculestrafficking inside plant cells, VOID is useful for calculating risksassociated with certain transformation or DNA delivery processes.

Since 1 out of 6 T-DNA molecules moved from the cytoplasm into thenucleus, 6 T-DNA molecules would be the minimal number of DNA moleculesthat need to be delivered into the cytoplasm per cell in order togenerate a transgenic plant containing one copy of the transgene in theplant genome. If 60 T-DNA molecules were delivered into cytoplasm percell, the risk of generating a transgenic plant containing multiplecopies of the transgene would increase 10 fold. Similarly, the risk ofgenerating a transgenic plant containing the transgene inserted at anundesired site would also increase 10 fold. If there were 600 T-DNAmolecules delivered into cytoplasm per cell, the risk of generating anundesired transgenic product would increase 100 fold, although thetransformation efficiency would also increase. One must compromisebetween transformation efficiency and the risk of an undesired outcome.The VOID procedure can facilitate the determination of an appropriatecompromise in the early stage of product development.

The risk associated with a particular gene delivery vehicle such as agene therapy vector or a DNA vaccine vector may be assessed in a similarfashion. For example, a gene therapy vector may be used to treat a testsubject. The number and location of the nucleic acid molecules insidethe treated cells may be determined as described above, at various timeintervals after the treatment. The ratio of the nucleic acid moleculespresent in the cytoplasm to those in the nucleus can be calculated. Therisk associated with the treatment can be then assessed as describedabove.

VI. Controlling the Copy Number of Nucleic Acid Molecules Delivered

During the development of a transgenic product or gene delivery vehicle,it is important to control the copy number of nucleic acid moleculesdelivered into target cells. In some circumstances, a high copy numberof the nucleic acid molecules may be desired in order to have a highexpression level of products encoded by the nucleic acid. In othercircumstances, only one copy of the nucleic acid molecule may be desiredper target cell. Since VOID can reveal the number and location ofnucleic acid molecules delivered during the early stage of development,VOID may be used to control the copy number of the nucleic acidmolecules. This may be achieved by manipulating the parametersassociated with the nucleic acid delivery, for instances, methods ofnucleic acid delivery, timing, length of time and conditions of thedelivery, and conditions (and types) of target cells.

In one embodiment, the number of T-DNA molecules delivered byAgrobacterium could be controlled by the length of time forco-cultivation. BY2 cells were cocultivated with LBA4404(pIG121-Hm) fordifferent time intervals. As shown in FIG. 4B, the T-DNA was barelydetectable in the plant cytoplasm at 2 h of cocultivation, which isconsistent with previous observations that T-DNA could be detected at 2h after infection of plants with A. tumefaciens (Virts & Gelvin. 1985.J. Bacteriol., 162, 1030-8; Narasimhulu et al. 1996. Plant Cell 8:873-886). Most of the T-DNA molecules entered the plant cytoplasm in 5 h(FIG. 4C). At 1 day of cocultivation, all the T-DNA molecules stillremained in the cytoplasm of the BY2 cells (FIG. 4D). At 2 days ofcocultivation, all the T-DNA molecules were inside the plant nuclei(FIG. 4E). This demonstrates that the number of T-DNA moleculesdelivered into cytoplasm can be controlled by choosing the appropriatelength of time for co-cultivation. Thus an appropriate copy number ofthe transgene in the target cell can be achieved.

In another embodiment, the number of T-DNA molecules delivered byAgrobacterium could be controlled by the use of different bacterialstrains to deliver the T-DNA molecules. When a virE2 mutant MX358(Stachel & Nester. 1986. EMBO J., 5, 1445-1454; Winans et al. 1987.Nucleic Acid Research 15: 825-836; Zupan et al. 2000. Plant J., 23,11-28) was used to conduct the cocultivation and VOID procedure, veryfew T-DNA molecules were found inside the BY2 cells (FIG. 2E). Bycontrast, when a wild-type Agrobacterium strain was used, some plantcells received hundreds of T-DNA molecules (FIG. 2A). This suggests thatmutation at virE2 may severely attenuate the bacterial ability todeliver the T-DNA into plant cells and demonstrates that DNA deliverydepends in part on the bacterial strains used.

The finding that virE2 can attenuate T-DNA delivery is consistent withthe recent evidence that the VirE2 protein can form a membrane channelto facilitate ssDNA transport (Dumas et al. 2001. Proc. Natl. Acad. Sci.USA, 98, 485-490). It may also indicate that T-DNA molecules werequickly degraded inside plant cells in the absence of the VirE2 protein,since VirE2 is known to bind to T-DNA and presumably can protect T-DNAfrom degradation (Zhu et al. 2000. J. Bacteriol., 182, 3885-3895; Zupanet al. 2000. Plant J., 23, 11-28).

While some plant cells received hundreds of T-DNA molecules when awild-type Agrobacterium strain was used (FIG. 2A), some receive only afew T-DNA (FIG. 3C). In a typical cocultivation experiment, the averagenumbers of T-DNA molecules inside the cytoplasm of a single BY2 cellthat received T-DNA were around 63. This demonstrates that DNA deliverydepends not only on the bacterial strains, but also on the target cells.In one embodiment, particular strains of Agrobacterium may be selectedfor a particular target cell population so that an appropriate number ofthe transgene is delivered into the cells.

VII. Screening for Molecular Markers Associated with Transformation andIdentifying VDI and Sec3 Homologs as Molecular Markers

In the context of this invention, molecular markers are proteins whichare required for, or assist in, the delivery of introduced DNA into thenucleus; thus molecular markers identify cells which are competent toreceive exogenous nucleic acid.

In identifying molecular markers associated with transformationcompetency, cells into which an exogenous nucleic acid has beenintroduced are monitored with VOID. The cells are further assayed forco-localization of a cellular protein with the exogenous nucleic acid todetermine whether the cellular protein is consistently in closeproximity to the exogenous nucleic acid. Co-localization would indicatethat the cellular protein is a molecular marker associated withcompetency to receive exogenous nucleic acid.

In one embodiment, the cellular protein and its location in the cell areidentified by immunohistology, using an antibody which bindsspecifically to the cellular protein. The antibody must be able to bindto the cellular protein (the antigen) in the fixed cell; usually, butnot always, this means the antibody must be able to bind to thedenatured form of the cellular protein.

In the context of this invention, the cellular protein is any proteinwhich is located in the cytoplasm, the cytosol, the plasma membrane, orthe nuclear membrane. Preferably the protein has a surface-exposed orcytoplasm-exposed domain if the protein is located in the plasmamembrane. Preferably the protein has a cytoplasm-exposed domain if theprotein is located in the nuclear membrane.

In a preferred embodiment, the cellular proteins to be screened as amolecular marker are those which interact with VirD2 or VirE2 (Gelvin.2000. Annu. Rev. Plant Physiol. Plant Mol. Biol., 51, 223-256). Inanother embodiment, the cellular protein is VIP1 (Tzfira et al. 2001.EMBO J., 20, 3596-3607), cyclophilins (Deng et al. 1998. Proc. Natl.Acad. Sci. USA, 95, 7040-7045), including specifically cyclophilinshaving Genbank accession numbers L14844, L14845 and U07276, AtKAPα(Ballas, N., Citovsky, V. 1997. Proc. Natl. Acad. Sci. USA, 94,10723-10728), and homologs of Sec3, including human, Drosophila, rat,mouse and Caenorhabditis elegans Sec3 (human exocyst component Sec3Accession number Q9NV70 and homolog accession number NP_(—)060731.1;Drosophila accession number Q9VVG4; rat accession number XP_(—)223340.1and XP_(—)223339.1; mouse accession number AAH24678.1; Caenorhabditiselegans accession number NP_(—)508530.1). In a another embodiment, thecellular protein is a protein of the Exocyst complex.

Immunohistology is performed according to methods known in the art (seefor example U.S. Pat. No. 5,869,274). In one method, a target antigenpresent in the sample is detected by a double antibody system. Initiallythe sample is incubated with a primary targeting antibody that isspecific for the target antigen. Detection of antigen-antibody complexescontaining the primary antibody and formed during the first incubationis accomplished by incubation with a second detecting antibody thatbinds to a region of the constant domain in the primary antibody; thesecond antibody is labeled. The result of the second incubation is, inthe presence of the target antigen, a complex of antigen and layers ofantibodies that contain the label.

In one embodiment, VOID is used to identify molecular markers associatedwith A. tumefaciens-mediated transformation of plant cells.

BY2 cells that had undergone co-cultivation with A. tumefaciens appearto be clustered in groups which either could, or could not, receiveT-DNA. Roughly half of the BY2 cells did not contain any T-DNAmolecules, suggesting that the competency of BY2 cells was important forBY2 cells to receive the T-DNA delivered by A. tumefaciens. When thecells received the T-DNA, often all the cells in the same clusterreceived T-DNA; otherwise, none of the cells in the same clusterreceived any T-DNA. The BY2 cells were undifferentiated; perhaps, thecell cycle stage may account for the competency to receive T-DNA. VOIDcould thus facilitate investigations on the plant cell competency toreceive T-DNA.

In order to screen for plant proteins that may be important forreceiving T-DNA molecules, the plant protein VDI (SEQ ID NO:1) thatinteracts with A. tumefaciens guide protein VirD2 was investigated as acandidate molecular marker by studying its position inside plant cellsduring T-DNA delivery. To localize VDI in A. thaliana and tobacco BY2cells, immunohistology was conducted with anti-VDI as the primaryantibody and anti-rabbit IgG-conjugated with Cy3 as the secondaryantibody. As shown in FIG. 5, VDI was localized in the cytoplasm of BY2(FIG. 5A) and A. thaliana cells (FIG. 5B), whereas no signal wasdetected in the control when preimmune serum instead of anti-VDI wasused (FIG. 5C).

Surprisingly, VDI was not expressed uniformly in all the plant cells.Some cells produced much more VDI than others; many cells did notproduce a detectable level of VDI proteins (FIG. 5). The BY2 cells wereundifferentiated; it is possible that the cell cycle stage may accountfor this phenomenon, as the individual cells are undergoing differentstages in cell cycling. This uneven expression of VDI in plant cells mayexplain why only certain cells rather than all BY2 cells could betransformed by A. tumefaciens (FIG. 6). In addition, this might berelated to why a high transformation efficiency is normally associatedwith freshly subcultured BY2 cells. Perhaps only these freshly dividedcells are competent to receive the T-DNA.

Double immunohistological staining of VDI and β-glucuronidase (GUS) wasconducted for Agrobacterium-transformed BY2 cells, using anti-VDI oranti-GUS as the primary antibody and anti-rabbit IgG conjugated-Cy3 oranti-mouse IgG conjugated-FITC as the secondary antibody. The sampleswere prepared after cocultivation of BY2 cells and preinduced A.tumefaciens for 3 days.

As shown in FIG. 7, the VDI (red dots) and reporter markerβ-glucuronidase (GUS) (green dots) coexisted in the same transformedcells. The BY2 cells producing VDI also expressed the GUS proteinencoded on the T-DNA delivered from Agrobacterium. In contrast, theremaining cells that lacked the VDI protein also did not produce anydetectable β-glucuronidase protein. This suggests that VDI is associatedwith Agrobacterium-mediated transformation of plants.

It is likely that only cells in a certain stage of the cell cycle canproduce VDI and consequently are competent to receive T-DNA andtransformed by Agrobacterium. Cells in other stage(s) could not produceVDI and consequently are not competent to receive T-DNA. This isconsistent with the observation that roughly half of undifferentiatedplant cells did not take up any T-DNA.

Coexistence of VDI with T-DNA was observed in transformed BY2 cells. Thesamples were prepared after coincubation of BY2 cells and preinducedLBA4404(pIG121-Hm) for 1 day. They were then subjected to the VOIDprocedure to observe T-DNA, and immunohistology to localize VDI. TheVOID procedure was performed with the same fragment of the GUS gene asthe probe, which was labeled with the Biotin High Prime kit; anti-Avidinwas used as the antibody to detect the T-DNA molecules (green dots inFIG. 8). Immunohistology was performed with anti-VDI and anti-rabbit IgGconjugated with Cy3 as the primary and secondary antibody, respectively.

As shown in FIG. 8, T-DNA molecules (green dots) appeared to coexistwith VDI (red dots) together in the plant cells transformed by A.tumefaciens. In contrast, no T-DNA molecules were detectable in thecells that did not produce VDI. This demonstrates that VDI played animportant role in the Agrobacterium-mediated plant transformation.

It was observed that VDI and T-DNA molecules were quite close to eachother (FIG. 8); this suggests that VDI may assist trafficking of theT-complex in the plant cytoplasm. As shown in FIGS. 5, 7 and 8, VDIprotein appeared to be randomly distributed in the cytoplasm ofuntransformed BY2 cells, while VDI was clustered with T-DNA in thetransformed BY2 cells at 1 day of cocultivation. After cocultivation for3 days, VDI protein became randomly distributed in the cytoplasm ofplant cells like the untransformed cells. In the control experiment, VDIwas still randomly distributed in the cytoplasm of BY2 cells, which werecocultivated with MX243 (virB mutant strain) that is unable to deliverT-DNA (Stachel, S. E., Nester, E. W. 1986. The genetic andtranscriptional organization of the vir region of the A6 Ti plasmid ofAgrobacterium tumefaciens. EMBO J., 5, 1445-1454) (FIG. 7B and FIG. 8B).These results clearly demonstrate that VDI actually participated in theprocess of Agrobacterium-mediated plant transformation.

The results described above indicate that VDI can be used as a molecularmarker for cell competency to receive T-DNA. Thus identification of VDIin a cell indicates that the cell is transformation competent.Expression of cellular VDI can be determined using known methods in theart, including immuno detection with anti-VDI antibodies, such asWestern blotting and ELISA, and immunoprecipitation of metabolicallylabeled cells using anti-VDI antibodies.

The plant VDI protein is homologous to the human Sec3 throughout theentire length of the proteins (see FIG. 9 for a comparison of VDI with anumber of exemplary Sec3 sequences). This indicates that VDI is anortholog of the human Sec3 protein. The Sec3 homologs are well conservedin eukaryotic cells. It is thus contemplated that homologs of Sec3,apart from VDI, also are useful as molecular markers for cell competencyto receive exogenous nucleic acid. Examples of Sec3 homologs includehuman Sec3 (Accession number Q9NV70 and NP_(—)060731.1), Drosophila Sec3(Accession number Q9VVG4), rat Sec3 (Accession number XP_(—)223340.1 andXP_(—)223339.1), mouse Sec3 (Accession number AAH24678.1), andCaenorhabditis elegans Sec3 (Accession number NP_(‘)508530.1).

Sec3 is a component of the Exocyst complex, which is well conserved ineukaryotic cells. Yeast and human Exocyst complexes have been identified(Matern et al. Proc Natl Acad Sci USA Aug. 14, 2001;98(17):9648-53).These Exocyst complexes exist as protein complexes consisting of severalprotein components. Since components of the Exocyst complex co-localizewith Sec3 protein (Matern et al. Proc Natl Acad Sci USA Aug. 14,2001;98(17):9648-53), it is contemplated that other components of theExocyst complexes may also be used as molecular markers of a cell'scompetency to receive exogenous nucleic acid.

VIII. Identifying, Characterizing and Producing Cells Competent toReceive Exogenous Nucleic Acid

It is contemplated that fusions of Sec3, including VDI, with GreenFluorescent Protein (GFP), is useful to identify, characterize andproduce cells competent to receive exogenous nucleic acid. It is furthercontemplated that regulated expression of exogenous Sec3 will producecells more competent to receive exogenous nucleic acid.

Since the Sec3 homolog VDI is correlated with transformation competence,competent cells can be selected if one is able to select for cellsexpressing Sec3 without killing the cells in the process. Methods forselecting cells expressing a particular protein are known in the art.For example, Sec3 protein may be fused in frame with GFP or itsvariants, and the fusion protein stably expressed in the cell under thecontrol of the native Sec3 promoter. Stable transformants expressingSec3-GFP can be directly isolated by fluorescence activated cell sorting(FACS) using appropriate excitation wavelengths and emission detector.Techniques for making the fusion constructs, stably introducing theconstructs into cells, and isolating and characterizing cells areroutinely practised in the art. In fact, a Sec3 homolog has been fusedwith GFP; the fusion protein has been expressed and correctly localizedin the cells (Matern et al. Proc Natl Acad Sci USA Aug. 14,2001;98(17):9648-53).

A variety of GFP mutants are available which have distinct spectralproperties, improved brightness and enhanced expression and folding inmammalian cells compared to the native GFP (Green Fluorescent Proteins,Chapter 2, pages 19 to 47, edited Sullivan and Kay, Academic Press, U.S.Pat. No. 5,625,048, U.S. Pat. No. 5,777,079, and U.S. Pat. No.5,804,387).

It is further contemplated that expression of Sec3 in cells that do notexpress endogenous Sec3 will enhance the cell's competence to receiveexogenous nucleic acid. As an example, Sec3 may be provided to a cell byway of an expression vector. The level of Sec3 may be regulated byplacing the gene encoding Sec3 under control of high expressioneukaryotic promoter/enhancers such as the CMV promoter/enhancer, SV40promoter/enhancer, RSV LTR,herpes simplex thymidine kinase promoter. Ina preferred embodiment, inducible promoters may be used to driveexpression of Sec3 so that Sec3 expression can be turned on only whenrequired, i.e. when nucleic acid delivery is carried out.

Inducible promoters contain transcription regulatory regions thatfunction to transcribe mRNA only when inducing conditions are present.Examples of suitable inducible promoters include the E. coli lacoperator responsive to IPTG, the metallothionein promotermetal-regulatory-elements responsive to heavy-metal (e.g. zinc)induction, the phage T7 lac promoter responsive to IPTG, the variousheat-shock promoters, the mouse mammary tumor virus (MMTV)steroid-inducible promoter, the synthetic GAL4-VP16 inducible system,Stratagene's LacSwitch™ inducible mammalian expression system,glucocorticoid response element containing promoter, and the ectdysonepromoter (U.S. Pat. No. 6,333,318).

EXAMPLES

Agrobacterium-Mediated Transformation

Tobacco (Nicoliana tabacum) BY2 suspension-cultured cells weremaintained in Murashige and Skoog (MS) liquid medium (Murashige, T., andSkoog, F. 1962. Physiol Plant 15: 473-497) supplemented with 0.2 mg/L of2,4-D; the cultures were incubated at RT with shaking at 100 rpm andsubcultured every week with a 4% inoculum. A. tumefaciens was grownovernight on AB medium; the cells were collected and then resuspended inIB medium (Cangelosi et al. 1991. Methods Enzymol., 204, 384-97)supplemented with 100 μM acetosyringone (AS). The cells were incubatedat 28° C. for 16-18 hr. After washing with MS medium, 100 μl of thebacterial cell suspension (5×10⁸ cells/ml) was added to 4 ml of BY2 cellsuspension that was 3 days old after the weekly subculturing. Afterincubation at RT for a certain time interval, the bacterial cells werewashed away from the plant cells as described previously (Lee et al.1999. J. Bacteriol. 181(1):186-196). The plant cells were then subjectedto the GUS assay or VOID.

VOID Monitoring of T-DNA in Transit

BY2 cells were subjected to the following VOID procedure after A.tumefaciens had transferred T-DNA into the BY2 cells. The cells werefixed in 2% paraformaldehyde for 2 h and then were washed for 3 timeswith a freshly prepared fixative solution (ethanol mixed with glacialacetic acid at a ratio of 3:1). They were kept in the fixative solutionat −20° C. until use.

The fixed cells were transferred to clean slides; the slides wereallowed to air-dry for 1-2 days at RT. Immediately before in situhybridization, the fixed cells on the slides were incubated in 0.2 %cellulase (in 0.01 M citrate buffer, pH 4.8) for 30 min at 37° C. Afterwashing for 3 times in 0.01 M citrate buffer for 10 min, the cells werepermeabilized with 0.2% Triton X-100 in PBS buffer for 10 min at 4° C.The cells were then washed for 3 times in PBS buffer for 10 min; theywere treated with 100 μg/ml of RNase A in 2×SSC for 60 min at 37° C.

After washing 3 times in 2×SSC for 5 min, the slides were dehydrated ina 70%, 90% and 100% ethanol series. After denaturation in hybridizationoven at 80° C. for 10 min, each slide was incubated with 20 μl of thehybridization mixture that had been heated at 75° C. for 10 min and thenchilled on ice for 10 min. The hybridization mixture contained 50%deionized formamide, 10% dextran sulfate, 2×SSC, 0.01% salmon sperm DNAand 10 ng/μl of a DNA probe that had been labeled withdigoxigenin-11-dUTP (DIG) using the DIG High Prime kit (RocheDiagnostics) with random primers and denatured at 95° C. for 10 min andchilled on ice for 10 min. The slides were then covered with a cleancoverslip and incubated overnight at 37° C. in a humid chamber.

After the coverslips were removed, the slides were washed twice in asolution containing 50% formamide and 2×SSC for 15 min at 37° C., washedonce at 37° C. in 2×SSC for 15 min, and then washed once at RT in 2×SSCfor 15 min, and finally washed once at RT for 5 min with PBS buffer. Theslides were incubated in a blocking solution (3% BSA in PBS buffer) at37° C. for 1 hr. To each slide was added 100 μl of rhodamine-conjugatedanti-DIG antibody (Roche Diagnostics) diluted (1:200) in the blockingsolution. The slide was covered with a coverslip and incubated in ahumid chamber at 37° C. for 45 min. After washing 4 times with 2×SSCcontaining 0.1% Tween-20 for 10 min, the slides were then dehydratedwith a 70%, 90% and 100% ethanol series. The slides were finallyair-dried and mounted with Vectashield mounting medium (Vectorlaboratories) containing PicoGreen (Molecular Probes) that cancounterstain the nuclei of BY2 cells.

The slides were examined with an Olympus Fluoview 300 confocalmicroscope system. The excitation light for the green and red signal was488 nm and 543 nm, respectively. The emission for the green and redsignal was 515-560 nm bandpass filter and 565 nm longpass filter,respectively. The images for the green and red signals were overlappedin a computer by using the software provided by Olympus.

Use of VOID to Identify Molecular Markers Associated with Transformation

To determine the subcellular location of a plant protein VDI (whichinteracts with Agrobacterium protein VirD2), the plant cells were fixedin 2% paraformaldehyde in 1×PBS/pH 7.4 for 3 hrs at room temperature.After cells were affixed to slides pre-coated with poly-L-lysine(Sigma), the slides were washed 3 times in 1×PBS for 10 min. After thecells were digested with 0.2% cellulase in 0.01 M citrate buffer (pH4.8) for 30 min at 37° C., they were permeabilized in 0.2% Triton X-100(in PBS) for 5 min at 4° C. The cells were washed 3 times in PBS for 10min and blocked in PBS containing 3% BSA for 1 hr. To each slide, 100 μlof primary antibody (anti-VDI) (diluted 1:100 into the blockingsolution) was then added. The slides were covered with a coverslip andincubated for 1 hr at RT in a humid chamber. After being washed 3 timeswith PBS containing 3% BSA for 10 min, the slides were incubated in 100μl of secondary antibody (FITC or CY3 conjugated) at a dilution of 1:100with a coverslip for 1 hr in a humid chamber at RT. The slides werewashed 3 times in PBS containing 0.1% Tween-20 for 10 min, then blowndry and mounted with a drop of Vectashield mounting medium (Vector Inc)with the coverslip sealed with clear nail polish to prevent drying andmovement under the microscope. In some cases, the mounting medium wassupplemented with PicoGreen (Molecular probes Inc) that can counterstainthe nuclei of plant cells.

The foregoing is considered as illustrative only of the principles ofthe invention. Since numerous modifications and changes will readilyoccur to those skilled in the art, it is not desired to limit theinvention to the exact modes of operation shown and described.Accordingly, all suitable modifications and equivalents may be resortedto, falling within the scope of the invention.

1. A process for monitoring exogenous nucleic acid in transit, thenucleic acid having been introduced into a cell, the process comprising:(a) providing a biological sample containing cells into which exogenousnucleic acid has been introduced, wherein the exogenous nucleic acid isin transit; (b) fixing the cells; and (c) subjecting the cells to an insitu hybridization procedure which comprises contacting thepermeabilized cells with a probe which hybridizes to the exogenousnucleic acid; and (d) visualizing the exogenous nucleic acid in transit.2. The process according to claim 1 for determining the number ofexogenous nucleic acid in the cytoplasm or in the nucleus.
 3. Theprocess according to claim 1 for determining whether the exogenousnucleic acid is in the cytoplasm or the nucleus.
 4. The processaccording to claim 1 for determining the length of time required for theexogenous nucleic acid to appear in the cytoplasm.
 5. The processaccording to claim 1 for determining the length of time required for theexogenous nucleic acid to appear from the cytoplasm to the nucleus. 6.The process according to claim 1 for determining the efficiency ofdelivery of the nucleic acid into the nucleus, the process furthercomprising the step of measuring the ratio of the number of theexogenous nucleic acid in the nucleus to the number of the exogenousnucleic acid in the cytoplasm.
 7. The process according to claim 1 forassessing risk associated with introduction of the exogenous nucleicacid into the cell, the process further comprising the step ofdetermining the number of exogenous nucleic acid in the cytoplasm and inthe nucleus at different time intervals after the exogenous nucleic acidhas been introduced; determining the ratio of exogenous nucleic acid inthe nucleus to cytoplasm at each interval; and predicting, in accordancewith said ratio and number of exogenous nucleic acid introduced, therisk associated with introduction of the exogenous nucleic acid into thecell.
 8. A process for determining the optimum parameters for obtaininga desired copy number of exogenous nucleic acid introduced into thecell, the process comprising: (a) introducing an exogenous nucleic acidinto a cell under a set of parameters; (b) monitoring the exogenousnucleic acid according to the process defined in claim 1 to determinethe number of exogenous nucleic acid in the cytoplasm or in the nucleusat different time intervals after the nucleic acid has been introduced;and (c) determining the set of parameters under which the exogenousnucleic acid is delivered in the desired copy number into the cell. 9.The process according to claim 8 wherein one of the parameters is thelength of time in which the exogenous nucleic acid is in contact withthe cell.
 10. The process according to claim 8 wherein one of theparameters is the ability of a gene delivery vector to deliver theexogenous nucleic acid.
 11. A process for determining the proportion ofcells competent to receive exogenous nucleic acid, the processcomprising: (a) introducing an exogenous nucleic acid to a portion of apopulation of cells; (b) monitoring the exogenous nucleic acid accordingto the process defined in claim 1 to determine the presence of theexogenous nucleic acid in the cell; and (c) determining the number ofcells in which the exogenous nucleic acid is present as a proportion ofthe portion of cells, wherein the proportion is the proportion of cellsof the population competent to receive the exogenous nucleic acid.
 12. Aprocess for identifying whether a cell contains an exogenous nucleicacid, wherein the exogenous nucleic acid is free of sequences encoding aselection marker or reporter protein intended to select for or identifythe cell as containing the exogenous nucleic acid, the processcomprising: (a) introducing the exogenous nucleic acid into the cell;and (b) monitoring the exogenous nucleic acid according to the processdefined in claim 1; wherein visualization of the nucleic acid in thecell indicates that the cell contains the exogenous nucleic acid.
 13. Aprocess for identifying a molecular marker associated with thecompetency of a cell to receive exogenous nucleic acid, wherein the cellcomprises an antigen, the process comprising: (a) introducing anexogenous nucleic acid to the cell; (b) monitoring the exogenous nucleicacid according to the process defined in claim 1; (c) testing the fixedcells for binding of the antigen with an antibody, wherein the antibodyis capable of binding to the antigen in the fixed and permeabilizedcell; and (d) determining whether the antigen co-localizes with theexogenous nucleic acid in transit; wherein co-localization of theexogenous nucleic acid in transit with the antigen indicates that theantigen is a molecular marker associated with transformation competency.14. A process for identifying a cell that is competent for receivingexogenous nucleic acid, the process comprising monitoring the exogenousnucleic acid according to the process defined in claim 1 for presence ofthe exogenous nucleic acid in the cell.
 15. The process according to anyone of claims 1 to 14 wherein the nucleic acid is DNA.
 16. The processaccording to claim 15 wherein the DNA is introduced into the cell byAgrobacterium.
 17. The process according to claim 1 wherein the in situhybridization procedure is fluorescence in situ hybridization.
 18. Theprocess according to claim 1 wherein the cell is a plant cell.
 19. Theprocess according to claim 18 further comprising the step of removingthe cell wall.
 20. The process according to claim 1, further comprisingthe step of permeabilizing the cells prior to contacting the cells withthe probe.
 21. A process for identifying a cell competent to receiveexogenous nucleic acid, the process comprising the step of identifyingexpression of a Sec3 protein in the cell, wherein Sec3 expressionindicates that the cell is competent to receive exogenous nucleic acid.22. The process according to claim 21, wherein the Sec3 protein isVirD2-Interacting protein (VDI), and wherein the cell is a plant cell.23. A process for identifying a cell competent to receive exogenousnucleic acid, comprising the step of identifying expression of acomponent of Exocyst complex in the cell, wherein expression of thecomponent indicates that the cell is competent to receive exogenousnucleic acid.
 24. The process according to 21 or 23 wherein the cell isa plant cell.
 25. A kit for monitoring exogenous nucleic acid intransit, the nucleic acid having been introduced into a cell, the kitcomprising: (a) reagents for fixing the cells; (b) reagents forpermeabilizing the fixed cells; (c) reagents for in situ hybridizationof a probe with the exogenous nucleic acid; and (d) instructions forusing the reagents (a) to (c) to monitor the exogenous nucleic acid intransit.
 26. A process for producing cells competent to receiveexogenous nucleic acid, the process comprising the step of expressingSec3 protein under control of an inducible promoter in the cell.
 27. Theprocess according to claim 26, wherein the Sec3 protein isVirD2-Interacting protein (VDI), and wherein the cell is a plant cell.